High-Throughput Microscopy Using Live Mammalian Cells
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INTRODUCTION Since the complete sequencing of several genomes, the systematic understanding of protein function and interaction has become one of the most important tasks in current molecular biology. Recent advances in light microscopy equipment regarding automation and acquisition speed offer the possibility to perform hundreds of experiments in parallel to study protein function and interaction in intact cells. In this context, live cell imaging is an excellent tool that may be used to investigate dynamic or rarely occurring cellular processes, to distinguish between primary and secondary phenotypes, and to study the phenotype kinetics. In this article, we present a guideline for high-throughput microscopy for functional screening in living cells. We shall examine each aspect of the general screening process and consider specific examples in the processing of time-lapse experiments. Although this article is largely based on examples from cultured mammalian cells, many of its considerations apply to cultured cells from other species such as Drosophila and to some extent to microscopy-based screening in small organisms such as Caenorhabditis elegans .Keywords:
High-Content Screening
Live cell imaging
High-Throughput Screening
Live cell imaging
Sample Preparation
Fluorescence-lifetime imaging microscopy
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Recent advancement in the area of green fluorescent protein techniques coupled with microscopic imaging has significantly contributed in defining and dissecting subcellular changes of apoptosis with high spatio-temporal resolution. Although single cell based studies using EGFP and associated techniques have provided valuable information of initiation and hierarchical changes of apoptosis, they are yet to be exploited for multiparameter cell based real time analysis for possible drug screening or pathway defining in a high throughput manner. Here we have developed multiple cancer cell lines expressing FRET sensors for active caspases and adapted them for high throughput live cell ratio imaging, enabling high content image based multiparameter analysis. Sensitivity of the system to detect live cell caspase activation was substantiated by confocal acceptor bleaching as well as wide field FRET imaging. Multiple caspase-specific activities of DEVDase, IETDase and LEHDase were analysed simultaneously with other decisive events of cell death. Through simultaneous analysis of caspase activation by FRET ratio change coupled with detection of mitochondrial membrane potential loss or superoxide generation, we identified several antitumor agents that induced caspase activation with or without membrane potential loss or superoxide generation. Also, cells that escaped the initial drug-induced caspase activation could be easily followed up for defining long term fate. Employing such a revisit imaging strategy of the same area, we have tracked the caspase surviving fractions with multiple drugs and its subsequent response to retreatment, revealing drug-dependent diverging fate of surviving cells. This thereby indicates towards a complex control of drug induced tumor resistance. The technique described here has wider application in both screening of compound libraries as well as in defining apoptotic pathways by linking multiple signaling to identify non-classical apoptosis inducing agents, the greatest advantage being that the high content information obtained are from individual cells rather than being population based.
High-Content Screening
Live cell imaging
Single-Cell Analysis
Fluorescence-lifetime imaging microscopy
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The development of preclinical models amenable to live animal bioactive compound screening is an attractive approach to discovering effective pharmacological therapies for disorders caused by misfolded and aggregation-prone proteins. In general, however, live animal drug screening is labor and resource intensive, and has been hampered by the lack of robust assay designs and high throughput work-flows. Based on their small size, tissue transparency and ease of cultivation, the use of C. elegans should obviate many of the technical impediments associated with live animal drug screening. Moreover, their genetic tractability and accomplished record for providing insights into the molecular and cellular basis of human disease, should make C. elegans an ideal model system for in vivo drug discovery campaigns. The goal of this study was to determine whether C. elegans could be adapted to high-throughput and high-content drug screening strategies analogous to those developed for cell-based systems. Using transgenic animals expressing fluorescently-tagged proteins, we first developed a high-quality, high-throughput work-flow utilizing an automated fluorescence microscopy platform with integrated image acquisition and data analysis modules to qualitatively assess different biological processes including, growth, tissue development, cell viability and autophagy. We next adapted this technology to conduct a small molecule screen and identified compounds that altered the intracellular accumulation of the human aggregation prone mutant that causes liver disease in α1-antitrypsin deficiency. This study provides powerful validation for advancement in preclinical drug discovery campaigns by screening live C. elegans modeling α1-antitrypsin deficiency and other complex disease phenotypes on high-content imaging platforms.
High-Content Screening
High-Throughput Screening
Phenotypic screening
Drug Development
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A Fully Automated High-Throughput Flow Cytometry Screening System Enabling Phenotypic Drug Discovery
The goal of high-throughput screening is to enable screening of compound libraries in an automated manner to identify quality starting points for optimization. This often involves screening a large diversity of compounds in an assay that preserves a connection to the disease pathology. Phenotypic screening is a powerful tool for drug identification, in that assays can be run without prior understanding of the target and with primary cells that closely mimic the therapeutic setting. Advanced automation and high-content imaging have enabled many complex assays, but these are still relatively slow and low throughput. To address this limitation, we have developed an automated workflow that is dedicated to processing complex phenotypic assays for flow cytometry. The system can achieve a throughput of 50,000 wells per day, resulting in a fully automated platform that enables robust phenotypic drug discovery. Over the past 5 years, this screening system has been used for a variety of drug discovery programs, across many disease areas, with many molecules advancing quickly into preclinical development and into the clinic. This report will highlight a diversity of approaches that automated flow cytometry has enabled for phenotypic drug discovery.
High-Content Screening
Phenotypic screening
High-Throughput Screening
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This chapter contains sections titled: Introduction Cell Lines for HTS Selection of the Most Suitable Cell Line Optimizing Cell Cultivation Adherence pH and Temperature Media and Additives Solvent Tolerance Cell Density Optimizing the Reproducibility of Seeding Signal Shift Edge Effect Cell Production and Plate Delivery The Amount of Cells Needed Cell Storage Conventional Cellular Screening Assays General HTS Assay Prerequisites Evaluation of Assay Quality ELISA-Based Assays Radiometric Cellular Assays Reporter Gene Assays Second Messenger Assays Ion Channel Assays The Definition of High-Content Screening Instrumentation for HCS Reagents (Fluorescent Probes) for HCS Low-Molecular-Weight Fluorophores Genetically Encoded Reporter for Fluorescence Detection Assays and Target-Based Applications of HCS GPCRs Kinases Other Drug Targets HCS Applications Targeting Generic Cellular Parameters and Morphology Outlook References
High-Content Screening
High-Throughput Screening
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Fluorescent live cell imaging has been widely used in biomedical studies since the introduction of the green fluorescent protein technology. However, many subcellular structures with sizes less than 200 nm are beyond the resolution limit of optical microscopes with visible light, therefore, super-resolution fluorescence microscopy attracts strong interest. In this review, the basic principles of several popular super-resolution fluorescence microscopy techniques are reviewed along with their biomedical applications especially live cell imaging.
Live cell imaging
Photoactivated localization microscopy
Fluorescence-lifetime imaging microscopy
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Immortalized cells, generated from two-dimensional cell culture techniques, are widely used in compound screening, lead optimization, and drug candidate selection. However, such cells lack many characteristics of cells in vivo. This could account for the high failure rates of lead candidates in clinical evaluation. New approaches from cell biology, materials science, and bioengineering are increasing the utility of three-dimensional (3D) culture. These approaches have become more compatible with automation and, thus, provide more physiologically relevant cells for high-throughput/high-content screening, notably in oncology drug discovery. Techniques range from simple 3D spheroids, comprising one or more cell types, to complex multitissue organoids cultured in extracellular matrix gels or microfabricated chips. Furthermore, each approach can be applied to stem cells, such as induced pluripotent stem cells, thereby providing additional phenotypic relevance and the exciting potential to enable screening in disease-specific cell types.
Phenotypic screening
High-Content Screening
High-Throughput Screening
Immortalised cell line
3D cell culture
Organoid
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Toxicity is a major cause of failure in drug discovery and development, and whilst robust toxicological testing occurs, efficiency could be improved if compounds with cytotoxic characteristics were identified during primary compound screening. The use of high-content imaging in primary screening is becoming more widespread, and by utilising phenotypic approaches it should be possible to incorporate cytotoxicity counter-screens into primary screens. Here we present a novel phenotypic assay that can be used as a counter-screen to identify compounds with adverse cellular effects. This assay has been developed using U2OS cells, the PerkinElmer Operetta high-content/high-throughput imaging system and Columbus image analysis software. In Columbus, algorithms were devised to identify changes in nuclear morphology, cell shape and proliferation using DAPI, TOTO-3 and phosphohistone H3 staining, respectively. The algorithms were developed and tested on cells treated with doxorubicin, taxol and nocodazole. The assay was then used to screen a novel, chemical library, rich in natural product-like molecules of over 300 compounds, 13.6% of which were identified as having adverse cellular effects. This assay provides a relatively cheap and rapid approach for identifying compounds with adverse cellular effects during screening assays, potentially reducing compound rejection due to toxicity in subsequent in vitro and in vivo assays.
High-Content Screening
Phenotypic screening
DAPI
High-Throughput Screening
Clonogenic assay
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Live cell imaging
Inverted microscope
Temporal resolution
Fluorescence-lifetime imaging microscopy
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Current optical microscope imaging heavily relies on fluorescence-based detection because of its high detection sensitivity and molecular specificity through labeling. However, the photophysics and photochemistry of the fluorophores, e.g., photobleaching, blinking and saturation, have limited its applicability to long-term observation and quantitative imaging. Label-free interference microscopy potentially overcomes the challenges aforementioned by measuring the scattered field of the sample quantitatively, but these approaches usually compromise on the sensitivity and molecular specificity of imaging. Here, we demonstrate coherent brightfield (COBRI) microscopy, a highly sensitive scatteringbased interference microscopy, to capture the dynamic linear scattering signal of live cell nucleus. We demonstrate a strategy to reconstruct the nuclear organization from the time-lapse COBRI imaging. Our method offers the opportunity to investigate the cell nuclear dynamics at the unprecedented spatial and temporal resolutions.
Photobleaching
Live cell imaging
Fluorescence-lifetime imaging microscopy
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